Project Summary Impaired endothelial function is observed in several disease states that are related to obesity, such as atherosclerosis, coronary artery disease, and diabetes. Reactive oxygen species (ROS) production and resultant oxidative stress contribute to the development of these obesity-related diseases. The enzyme NADPH oxidase is a major source of oxidative stress within the vasculature, and has been linked with the Metabolic Syndrome. In our previously funded studies, we demonstrated for the first time that: 1) in vivo ROS were elevated in skeletal muscle of obese as compared to lean or overweight human subjects, 2) perfusion of the NADPH oxidase inhibitor apocynin locally into muscle normalized ROS levels and reversed local microvascular endothelial dysfunction in the obese individuals, and 3) aerobic exercise training was effective at attenuating in vivo H2O2 production and reversing microvascular endothelial dysfunction in the obese individuals. However, the mechanism of these findings, particularly with respect to the specific sources of ROS, is not clear. We will investigate in this R15 renewal application the mechanism of exercise training-induced alterations in ROS production and action on endothelial dysfunction in obesity using our newly developed microdialysis methodology of monitoring ROS production, in combination with analysis of muscle biopsy samples obtained before and after our previously tested 8-week intervention of aerobic interval exercise training. The objectives of this study are to determine the impact of in vivo NADPH oxidase activity on endothelial function in obese individuals, and to determine the mechanism of training-induced improvements in endothelial function. Our unique microdialysis methodology will allow monitoring of microvascular/endothelial function and ROS generation, as well as the administration of pharmacological agents directly into muscle. The central hypothesis is that it is upregulation of both mitochondrial ROS and NADPH oxidase-derived ROS that results in endothelial dysfunction in obesity, and that exercise training down-regulates mitochondrial-derived ROS, and NADPH oxidase 4, thereby improving endothelial function. The aims of this proposal are to: 1) determine the contributions of mitochondrial ROS and specific NADPH oxidase isoforms to the NADPH oxidase dependent endothelial dysfunction in skeletal muscle of obese individuals; 2) determine the mechanism of ROS reduction and improved endothelial function resulting from an 8-week aerobic interval training program.